2-methyl-3-butenyl-1-pyrophosphoric acid salts and agents for treating lymphocytes

ABSTRACT

A pharmaceutically acceptable salt of 2-methyl-3-butenyl-1-pyrophosphoric acid; an agent for treating lymphocytes which comprises at least one of 2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptable salt thereof, and a hydrate thereof; Vγ2Vδ2 type T cells treated by the same; and a medicine containing the same specifically stimulate and proliferate the human Vγ2Vδ2 type T cells, and also induce and enhance an antitumor activity thereof.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP00/00273 which has an Internationalfiling date of Jan. 21, 2000, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a novel salt of an organicpyrophosphoric acid compound. It also relates to an agent for treatinglymphocytes containing the organic pyrophosphoric acid compound or thesalt thereof which effectively induces and potentiates an antitumoreffect, Vγ2Vδ2 type T cells treated by the agent for treatinglymphocytes and to a medicine containing the same.

BACKGROUND ART

As a method for inducing and enhancing an antitumor effect in humanlymphocytes, it has been known LAK therapy using interleukin-2. That is,it has been known that by having about 800 U/ml of interleukin-2 to acton the lymphocytes, cell groups induced thereby with an antitumoractivity can be used as an antitumor effecter. However, in this method,there are serious side effects, for example, destruction of the selfcells such as endovascular cells due to non-specific cytotoxic propertypossessed by LAK cells and the like or induction of autoimmunity due tonon-specific activation of T cells by interleukin-2, thereby making itdifficult to apply this methods to an actual clinical field.

As compounds which specifically activate Vγ2Vδ2 type T cells, there havebeen known mycobacteria-derived isopentenyl pyrophosphoric acid and monoethyl phosphoric acid obtained by an organic synthesis. In thosemethods, however, the concentrations of those compounds are required tobe several hundreds of μM to several mM in order to activate Vγ2Vδ2 typeT cells. Such high concentrations of the compounds may have a toxiceffect on the cells, therefore, it was difficult to use those compoundsto induce and enhance an antitumor effect of the lymphocytes in a largescale. In any case, it has not yet been known a synthetic compound whichcan act on the Vγ2Vδ2 type T cells in a concentration of severalhundreds of nM to several hundreds of μM, and specifically proliferatethose cell groups.

The present invention has been aimed to solve the above-mentionedproblems of the prior art. An object of the present invention is toprovide a novel compound that can specifically stimulate and proliferatethe human Vγ2Vδ2 type T cells, an agent for treating lymphocytes thatinduces and/or potentiates an antitumor effect of the human Vγ2Vδ2 typeT cells, Vγ2Vδ2 type T cells treated by the same, and a medicine whichcomprises the same.

SUMMARY OF THE INVENTION

The present inventors have made extensive and intensive studies insearch for a compound that can specifically stimulate and proliferatethe human Vγ2Vδ2 type T cells thereby inducing and enhancing theantitumor effect thereof. As a result, they have found apharmaceutically acceptable salt of 2-methyl-3-butenyl-1-pyrophosphoricacid, especially a sodium salt thereof as a novel organic pyrophosphoricacid compound. That is, they have found that, when lymphocytes in humanblood such as peripheral blood or the lymph are treated by such anorganic pyrophosphoric acid compound, Vγ2Vδ2 type T cells arespecifically stimulated and proliferated whereby the antitumor effectthereof are induced and potentiated, and thus, the present invention hasbeen completed.

Accordingly, the present invention relates to a pharmaceuticallyacceptable salt of 2-methyl-3-butenyl-1-pyrophosphoric acid which is anovel organic pyrophosphoric acid compound, especially a sodium saltthereof and to an agent for treating lymphocytes which comprises atleast one selected from the group consisting of2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptablesalt thereof, especially a sodium salt thereof, and a hydrate thereof.Further, the present invention relates to Vγ2Vδ2 type T cells treated bythe agent for treating lymphocytes, and a medicine which comprises thesame.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram showing an elution curve of the sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid synthesized in Example by Qsepharose HP column chromatography using 820 nm absorption as aparameter.

FIG. 2 is a graph showing a relation of a specific lysis ratio relativeto an E/T ratio when Vγ2Vδ2 type T cells treated in Test 1 acted onDaudi cells.

FIG. 3 is a graph showing a relation of a specific lysis ratio relativeto an E/T ratio when Vγ2Vδ2 type T cells treated in Test example 2 actedon EJ-1 cells.

FIG. 4 is a graph showing a relation of a specific lysis ratio relativeto an E/T ratio when Vγ2Vδ2 type T cells treated in Test example 3 actedon T24 cells.

FIG. 5 is a graph showing a relation of a specific lysis ratio relativeto an E/T ratio when Vγ2Vδ2 type T cells treated in Test example 4,subsequently frozen for preservation and thawed, acted on Daudi cells.

FIG. 6 is a graph showing a relation of a specific lysis ratio relativeto an E/T ratio when Vγ2Vδ2 type T cells treated in Test example 5,subsequently frozen for preservation and thawed, acted on EJ-1cells.

FIG. 7 is a graph showing a relation of a specific lysis ratio relativeto an E/T ratio when Vγ2Vδ2 type T cells treated in Test example 6,subsequently frozen for preservation and thawed, acted on T 24 cells.

FIG. 8 is a graph showing a relation of a specific lysis ratio relativeto an E/T ratio when each of the Vγ2Vδ2 type T cells collected from theperipheral blood of A, treated, frozen for preservation and thawed andVγ2Vδ2 type T cells collected from the peripheral blood of F and treatedrespectively acted on Daudi cells.

FIG. 9 is a graph showing a relation of a specific lysis ratio relativeto an E/T ratio when each of the Vγ2Vδ2 type T cells collected from theperipheral blood of A, treated, frozen for preservation and thawed andVγ2Vδ2 type T cells collected from the peripheral blood of F and treatedrespectively acted on normal peripheral blood cells of A.

BEST MODE FOR CARRYING OUT THE INVENTION

The novel pyrophosphoric acid compound of the present invention is apharmaceutically acceptable salt of 2-methyl-3-butenyl-1-pyrophosphoricacid, and sodium salt is a representative. The sodium salt isrepresented by the formula (I):

(wherein each of X independently represents a hydrogen atom or a sodiumatom, at least one of which is a sodium atom). It is preferable that 2of X are sodium atoms due to the compatibility to in vivo cells.Further, it can contain water of crystallization. As a pharmaceuticallyacceptable salt, there may be mentioned a potassium salt, an ammoniumsalt, a triethyl ammonium salt and an amino acid salt such as lysinesalt in addition to the above-described sodium salt.

2-Methyl-3-butenyl-1-pyrophosphoric acid sodium salt of the presentinvention can be synthesized, for example, in a method described asfollows. That is, 2-methyl-3-butenyl-1-ol is reacted in a solvent suchas acetonitrile with bis(triethyl ammonium)phosphoric acid usingtrichloroacetonitrile as a catalyst. The obtained reaction product isseparated with diethyl ether and an aqueous ammonia solution, then, theproduct extracted in the diethyl ether layer is applied to an anionexchange column chromatography, followed by elution from triethylammonium bicarbonate buffer by a concentration radient method, to obtain2-methyl-3-butenyl-1-pyrophosphoric acid. Subsequently, it is treatedwith a Na type cation exchange resin to obtain a sodium salt. Otherpharmaceutically acceptable salts can be synthesized according to theabove method.

The thus obtained sodium salt of 2-methyl-3-butenyl-1-pyrophosphoricacid is white powder with deliquenscence, which becomes a white viscoussubstance when it contains water. When it is dissolved in a neutralaqueous solution, for example, at pH 6 to 7, it can be stably preservedmore than one week at room temperature, and more than one year at −20°C. However, in an acidic range of pH 4 or less, dissociation ofinorganic pyrophosphoric acid is observed within a few minutes.

2-Methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptablesalt thereof, especially a sodium salt thereof and a hydrate thereofspecifically stimulate and proliferate the human Vγ2Vδ2 type T cellsexisting in the human blood such as the peripheral blood or the humanlymph, and at the same time, they promote induction and enhancement ofantitumor activity of those cells. As the pharmaceutically acceptablesalt, the above-mentioned salts are exemplified.

Accordingly, when the human blood or lymph is treated with an agent fortreating lymphocytes of the present invention containing at least onekind of the above mentioned organic pyrophosphoric acid derivatives, itspecifically stimulates and proliferates the human Vγ2Vδ2 type T cells,and at the same time, it can induce and enhance the antitumor activitythereof. In this case, an analysis using the human Vγ2Vδ2 type T cellclones shows that 2-methyl-3-butenyl-1-organic pyrophosphoric acidcompound, especially the above described novel sodium salt can stimulatethe cells even in a very small amount such as several hundreds nM toseveral tens μM, and proliferate the human Vγ2Vδ2 type T cells in thepresence of a very small amount of several hundreds μM, thereby inducingand enhancing the antitumor activity thereof.

As an organic pyrophosphoric acid compound to be contained as an activecomponent in the agent for treating lymphocytes of the presentinvention, it is exemplified by the above-mentioned2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptablesalt thereof and a hydrate thereof. Among those, a sodium salt ispreferable due to a good compatibility to in vivo cells, and a sodiumsalt wherein 2 of X are sodium atoms in the above-mentioned formula (I)is especially preferable. Those salts can be used in combination withouta need for isolation, in a proper pH range to meet the requirements,preferably in a range of pH 6 to 7

When the agent for treating lymphocytes containing the above-mentionedorganic pyrophosphoric acid compound is acted on the human blood,especially on the peripheral blood, if interleukin-2 is added as acofactor in a concentration of 1 to 20 U/ml, specific proliferation ofthe Vγ2Vδ2 type T cells becomes outstanding. As long as theconcentration of the interleukin-2 is limited under 20 U/ml,non-specific activation of the lymphocytes would not occur differentfrom the case of the LAK cells. Further, it is possible to use othercofactor such as interleukin-15. The human Vγ2Vδ2 type T cells have akind of natural killer activity. The present invention is completelydifferent from conventional induction methods of the antitumor activityin that it can proliferate those cell groups antigen-specifically.

That is, in the LAK therapy and the TIL therapy, the cell proliferationfactor such as intrerleukin-2 is added excessively thereby forciblyactivating naive cell groups in the peripheral blood. Therefore, it isaccompanied by side effects such as attacking the self cells. On thecontrary, the above-mentioned organic pyrophosphoric acid compound isnaturally to be used to proliferate the population of the Vγ2Vδ2 type Tcells having natural killer activity, therefore, it is not excessivelyand forcibly enhancing the antitumor activity by using an excess amountof the antigen. The concentration of 20 μM of the above-mentionedorganic pyrophosphoric acid type antigen corresponds to the activity of10 to 50 U, and is appropriate as a concentration. Therefore,proliferation and activation of the human Vγ2Vδ2 type T cells andinduction and enhancement of the antitumor activity thereof by thepresent invention is immunochemically appropriate causing no destructionof the self cells. From the above, the agent for treating lymphocyte ofthe present invention is a more advantageous agent for inducing andenhancing the antitumor activity due to its higher specificity and lessside effects in comparison to the LAK therapy and the TIL therapy.

In addition, the use of the agent for treating lymphocytes of thepresent invention is advantageous in that it is possible to freeze thetreated cells. That is, the blood containing lymphocytes and/or thelymph are collected at the suitable point and preserved by freezing thenas necessity arises, the lymphocytes are activated by theabove-mentioned organic pyrophosphoric acid compound. By proliferatingthe Vγ2Vδ2 type T cells as mentioned above, and by inducing andenhancing the antitumor activity thereof, it is possible to decrease aburden for donors of peripheral blood lymphocytes.

Further, since the human Vγ2Vδ2 type T cells have natural killeractivity, even if it is preserved by freezing after the antitumoractivity is induced and potentiated, it can exhibit the antitumoractivity immediately after it thaws. This makes it possible to use thecell at any time. On the contrary, the LAK therapy and the TIL therapyrequire fresh cells all the time, and every time the antitumor activityis induced and potentiated, the lymphocytes should be collected and theproliferation factor should be added. From the above-mentioned facts,the therapy using the agent for treating lymphocytes of the presentinvention is clearly more advantageous than the LAK therapy and the TILtherapy. Therefore, the present invention enables the wide range ofantitumor therapy in the practical medical field by using a medicinewhich comprises the human Vγ2Vδ2 type T cells whose antitumor activityis induced and potentiated.

The Vγ2Vδ2 type T cells which are treated and proliferated with theagent for treating lymphocytes of the present invention, and whoseantitumor activity is induced and potentiated can exhibit the antitumoractivity by returning those T cells themselves into the human patient asthe peripheral blood. The administration method includes a localinjection, intravenous injection, endermic absorption and the like.

The thus treated Vγ2Vδ2 type T cells are free from MHC limitation and itis possible to administer them to the other person.

Therefore, the present invention includes the Vγ2Vδ2 type T cells whichare treated with the agent for treating lymphocytes of the presentinvention and a medicine containing the same.

When the compound of the present invention or the agent for treatinglymphocytes containing the same is administered as a medicine, it can beprepared by a general preparation technique, and it can be used as asolid or liquid preparation form such as a tablet, a capsule, powders,granules, a suppository, cream, an ointment, an aqueous solution, anemulsion, oil or a suspension and the like.

Further, in that case, except for preparations whose pH is 4 or less,generally used additives in the preparation can be used as necessityarises, such as an excipient, a disintegrator, a lubricant, a binder, apreservative, a stabilizer, an osmotic pressure adjuster, a substrateand the like.

The examples of the above-mentioned additives include glucose, lactose,starch, carboxymethyl cellulose, magnesium stearate, talc, liquidparaffin, polyvinyl alcohol, vegetable oil, polyalkylene glycol, and thelike. It can also contain a medical component in addition to these.

EXAMPLES

The present invention will be explained in more detail referring toExamples and Test examples hereinbelow. These examples, however, are notconstrued to limit the scope of the present invention.

In Test examples, treatment of the peripheral blood by2-methyl-3-butenyl-1-pyrophosphoric acid or by a salt thereof wasconducted as follows unless otherwise specified. Lymphocytes containedin the peripheral blood derived from healthy volunteer were treated sothat the final concentration of the pyrophosphoric acid compound was 20μM, and cultured in a Yssel's medium under conditions of temperature at37° C. and a carbon dioxide concentration of 5%. After 2, 4, 6, 8 and 10days, interleukin-2 was added in an amount of 10 U/ml, respectively, asa cofactor. After 12 days, the cells were collected.

EXAMPLE Synthesis of Sodium Salt of 2-Methyl-3-butenyl-1-pyrophosphoricAcid

To 1 mol of 2-methyl-3-butenyl-1-ol was added 1 mol oftrichloroacetonitrile as a catalyst, and while stirring at a temperatureof 25° C., 4 mol of bis (triethyl ammonium)phosphoric acid dissolved in20 ml of acetonitrile was added dropwise over 4 hours and reacted whilestirring for another 2 hours. To the reaction product was added 100 mlof diethyl ether and the mixture was transferred to a separation funnel.100 ml of 0.88% aqueous ammonia was added thereto and shaken, and theobtained organic pyrophosphoric acid was extracted in an organic layer.After diethyl ether was evaporated from the organic layer under areduced pressure, the resultant liquid was subjected to Q Sepharose HPanion exchange column chromatography having a diameter of 2.5 cm and alength of 8 cm, then washed with water.

Subsequently, using a triethyl ammonium bicarbonate buffer, the organicpyrophosphoric acid compound was eluted by a concentration gradientmethod from 0 to 500 mM. An elution curve of the column chromatographywith absorption at 820 nm as a parameter is shown in FIG. 1. Here, afraction eluted in an area of approximately 150 to 200 mM was2-methyl-3-butenyl-1-pyrophosphoric acid. After freeze-dried thisfraction, it was dissolved in 2 ml of water and the solution was treatedby Na type Dowex 50 W cation exchange resin (Trade name of Dow ChemicalCo.) to obtain a sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidcontaining a little amount of water as a white viscous substance. It wasdried to obtain a sodium salt of 2-methyl-3-butenyl-1-pyrophosphoricacid as white powder.

TEST EXAMPLE 1

Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells andinduction and enhancement by the treatment of the human peripheral bloodderived from healthy volunteers A, B and C were conducted according tothe above-mentioned method. The peripheral blood containing thepotentiated human Vγ2Vδ2 type T cells were acted on Daudi cells toanalyze cytotoxicity. The results are shown in FIG. 2. In FIG. 2, thetransverse axis shows an effecter/target ratio (hereinafter refer to asan E/T ratio), that is, a ratio of existing human Vγ2Vδ2 type T cellsand Daudi cells. The vertical axis shows a specific lysis ratio.

As is apparent from FIG. 2, in both case of the peripheral blood of Aand B, cytotoxicity reached a plateau even when the E/T ratio is 1:1,exhibiting a high cytotoxic effect. In the case of the peripheral bloodof C, cytotoxicity reached the same level when the E/T ratio is 5:1.

TEST EXAMPLE 2

Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells andinduction and enhancement by treatment of the human peripheral bloodderived from healthy volunteers A, B and C were conducted in the samemanner as in Test example 1 according to the above-mentioned method. Theperipheral blood containing the potentiated human Vγ2Vδ2 type T cellswere acted on EJ-1 cells to analyze cytotoxicity. The results are shownin FIG. 3. In FIG. 3, the transverse axis shows an E/T ratio, and thevertical axis shows a specific lysis ratio.

As is apparent from FIG. 3, in both cases of the peripheral blood of Aand B, cytotoxicity reached a plateau when the E/T ratio is 5:1, and incase of the peripheral blood of C, cytotoxicity reached approximately aplateau when the E/T ratio is 10:1.

TEST EXAMPLE 3

Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells andinduction and enhancement by treatment of the human peripheral bloodwere conducted according to the above-mentioned method. The peripheralblood containing the potentiated human Vγ2Vδ2 type T cells were acted onT24 cells to analyze cytotoxicity. The results are shown in FIG. 4. InFIG. 4, the transverse axis shows an E/T ratio, and the vertical axisshows a specific lysis ratio.

As is apparent from FIG. 4, in all cases of the peripheral bloods of A,B and C, cytotoxicity reached approximately a plateau when the E/T ratiois 20:1.

TEST EXAMPLE 4

Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells andinduction and enhancement by treatment of the human peripheral bloodwere conducted according to the above-mentioned method using theperipheral blood of the volunteer A and other healthy volunteers D andE. Thus obtained treated peripheral blood was preserved by freezing forone year at −20° C. The treated and preserved blood after being thawedwere acted on Daudi cells to analyze cytotoxicity. The results are shownin FIG. 5. In FIG. 5, the transverse axis shows an E/T ratio, and thevertical axis shows a specific lysis ratio.

As is shown by FIG. 5, in both cases of the peripheral bloods of A andE, cytotoxicity reached approximately a plateau when the E/T ratio is20:1, and in a case of the frozen-preserved peripheral blood of D,cytotoxicity reached a plateau when the E/T ratio is 20:1 while there isa difference in degree. As a result, it is evident that the treatedlymphocytes maintain sufficient antitumor effect even after they arepreserved by freezing.

TEST EXAMPLE 5

Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells bytreatment of the human peripheral blood and induction and enhancement aswell as preservation by freezing and thawing were conducted in the samemanner as in Test example 4. The treated and preserved blood was actedon the EJ-1 cells to analyze cytotoxicity. The results are shown in FIG.6. In FIG. 6, the transverse axis shows an E/T ratio, and the verticalaxis shows a specific lysis ratio.

As is apparent from FIG. 6, cytotoxicity reached approximately a plateauwhen the E/T ratio is 20:1 in any case while there is a difference indegree. And it is observed that the antitumor effect was maintained evenafter the cells are preserved by freezing.

TEST EXAMPLE 6

Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Example, proliferation of the human Vγ2Vδ2 type T cells bytreatment of the human peripheral blood and induction and enhancement aswell as preservation by freezing and thawing were conducted in the samemanner as in Test example 4. The treated and preserved blood was actedon the T24 cells to analyze cytotoxicity. The results are shown in FIG.7. In FIG. 7, the transverse axis shows an E/T ratio, and the verticalaxis shows a specific lysis ratio.

As is apparent from FIG. 7, cytotoxicity reached approximately a plateauwhen the E/T ratio is 20:1 in any case while there is a difference indegree. And it is observed that the antitumor effect was maintained evenafter the cells were preserved by freezing.

TEST EXAMPLE 7

Using the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoric acidobtained in Test example and using the peripheral blood of the volunteerA, proliferation of the human Vγ2Vδ2 type T cells by treatment of thehuman peripheral blood and induction and enhancement were conductedaccording to the above-mentioned method. In the same manner as in Testexample 4, the treated and preserved blood which had been preserved byfreezing and thawed were acted on Daudi cells to analyze cytotoxicity.On the other hand, peripheral blood of other healthy volunteer F wastreated according to the above-mentioned method and they were acted onDaudi cells in the same manner to analyze cytotoxicity. The results areshown in FIG. 8. Further, using the treated and preserved blood derivedfrom A that had been preserved by freezing and thawed and the treatedperipheral blood derived from F, cytotoxicity was analyzed based on thenormal peripheral blood cells of A. The results are shown in FIG. 9. Inthose FIGS., the transverse axis shows an E/T ratio, and the verticalaxis shows a specific lysis ratio.

As is apparent from FIG. 8, cytotoxicity reached approximately a plateauwhen E/T ratio is 20:1 in both cases of the peripheral bloods of A(after preservation by freezing and thawing) and F.

On the other hand, from the results for the peripheral bloods of A(after preservation by freezing and thawing) and F in FIG. 9, it isevident that the Vγ2Vδ2 type T cells whose antitumor activity wasinduced and potentiated by the sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid do not attack the normal cellswhen they are used as they are or even after they are preserved byfreezing and thawed.

TEST EXAMPLE 8

10 ml of the peripheral blood collected from a healthy volunteer G wassubjected to Ficoll-Paque specific gravity centrifugation to purify theperipheral blood mononuclear cells and they were suspended in a Yssel'smedium. The cells were apportioned into a 24-hole plate so that thenumber of the cells becomes a concentration of 2.5 million/1.5 ml/hole,and then, according to the above-mentioned method, treatments 1 to 5were conducted as shown in Table 1. Among them, treatment 1 is atreatment using only a culture medium and treatment 2 solely usesinterleukin-2, and both treatments are for comparison. Treatments 3 to 5are treatments of the present invention using the sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid obtained in Example andinterleukin-2 in combination. On the day 11 after conducting thetreatment, a ratio of Vγ2Vδ2 type T cells in existing CD3 cells wascalculated. The results are shown in Table 1.

TABLE 1 Treatment Sodium salt of 2- V_(γ)Vδ2 type Treat-methyl-3-butenyl- T cells/ ment 1-pyrophosphoric Interleukin-2 CD3 cellsNo. acid [μM] [U/ml] [%]  1* — — 6.7  2* — 10 6.9 3 2 10 58.3 4 20 1085.5 5 200 10 94.5 *: Comparative treatment

As is apparent from Table 1, when interleukin-2 was used solely, theratio of Vγ2Vδ2 type T cells was approximately the same level as that ofthe treatment using only the culture medium. On the contrary, in thetreatments 3 to 5 of the present invention, proliferation of the Vγ2Vδ2type T cells by the sodium salt of 2-methyl-3-butenyl-1-pyrophosphoricacid was significant and the ratios were increased accompanying anincrease of the amount of the pyrophosphoric acid compound added.

Industrial Applicability

As is explained above, 2-methyl-3-butenyl-1-pyrophosphoric acid, apharmaceutically acceptable salt thereof, especially the sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid of the present invention and/oran agent for treating lymphocytes containing a hydrate thereof as anactive component stimulate and proliferate the human Vγ2Vδ2 type Tcells, and at the same time, exhibit a specific effect to induce andenhance the antitumor effect thereof. In addition, they do not exhibitany cytotoxicity on the self cells. They can specifically activate anatural killer cell such as the human Vγ2Vδ2 type T cell with a lowantigen concentration.

The agent for treating lymphocytes of the present invention can beapplied to various clinical fields. For example, peripheral blood of acancer patient or a healthy person is collected and treated by the agentfor treating lymphocytes of the present invention to stimulate Vγ2Vδ2type T cells, thereby proliferating them polyclonally as well asinducing and enhancing the antitumor activity thereof. By re-circulatingthose lymphocytes in the body, the antitumor activity in vivo can beexpected. In that case, it is possible to preserve the lymphocytes byfreezing, they can be frozen and preserved at a suitable stage, and canbe administered to the patient as a need arises.

What is claimed is:
 1. A pharmaceutically acceptable salt of2-methyl-3-butenyl-1-pyrophosphoric acid.
 2. A sodium salt of2-methyl-3-butenyl-1-pyrophosphoric acid represented by the formula (I):

wherein each of X independently represents a hydrogen atom or a sodiumatom, at least one of which is a sodium atom.
 3. The sodium saltaccording to claim 2, wherein two of X are sodium atoms.
 4. An agent fortreating lymphocytes which comprises at least one of2-methyl-3-butenyl-1-pyrophosphoric acid, a pharmaceutically acceptablesalt thereof, on a hydrate thereof.
 5. The agent for treatinglymphocytes according to claim 4, wherein the pharmaceuticallyacceptable salt of 2-methyl-3-butenyl-1-pyrophosphoric acid is a sodiumsalt of 2-methyl-3-butenyl-1-pyrophosphoric acid.
 6. The agent fortreating lymphocytes according to claim 4, wherein the agent furthercomprises interleukin-2.
 7. The agent for treating lymphocytes accordingto claim 5, wherein the agent further comprises interleukin-2.